Mobile phase for HPLC

[esbena]

Hi everyone..

I´m new to HPLC and trying to prepare a mobile phase for creatinine quantification in mice..

I need some help with my calculations of the relative amounts of the different components

MilliQ (96.6) : Acetonitrile (3) : Trifluoroacid (0.1)

in a total of 500 ml

Thanks

[PeterS]

Well

Do you mean 96.9 instead of 96.6?

In that case 484.5 ml MilliQ, 15 ml Acetonitril, 0.5 ml acetonitril. What column do you use since creatinine has a low affionity for C18 HPLC columns.

PeterS

[HPLCaddict]

I've got my pedantic 5 minutes right now : 484.5 mL H20 + 15 mL ACN + 0.5 mL TFA will NOT give you a total volume of 500 mL.
BTW how do you measure 484.5 mL H2O volumetrically?

To the OP: Where did you get that mobile phase recipe from? Isn't there a decent decription on how to prepare it? Are you in a regulated environment?
Basically, this sounds like water/ACN 97:3 with 0.1% TFA. In absence of a decent recipe I'd mix 970 mL water with 30mL ACN and add 1mL TFA.
And yes, I wouldn't care if it's 970 or 969mL .
And yes, I'd be aware that the one who developed that method might have prepared his mobile phase in a different way. If the method's so sensitive (aka not robust) that you get severe chromatographic differences with mobile phases of 97:3 or 96:3 or 98:3 composition, it's not worth using it...